Bazedoxifene does not share estrogens effects on IgG sialylation

The incidence of rheumatoid arthritis (RA) increases at the same time as menopause when estrogen level decreases. Estrogen treatment is known to reduce the IgG pathogenicity by increasing the sialylation grade on the terminal glycan chain of the Fc domain, inhibiting the binding ability to the Fc gamma receptor. Therefore, treatment with estrogen may be beneficial in pre-RA patients who have autoantibodies and are prone to get an autoimmune disease. However, estrogen treatment is associated with negative side effects, therefore selective estrogen receptor modulators (SERMs) have been developed that have estrogenic protective effects with minimal side effects. In the present study, we investigated the impact of the SERM bazedoxifene on IgG sialylation as well as on total serum protein sialylation. C57BL6 mice were ovariectomized to simulate postmenopausal status, followed by ovalbumin immunization, and then treated with estrogen (estradiol), bazedoxifene, or vehicle. We found that estrogen treatment enhanced IgG levels and had a limited effect on IgG sialylation. Treatment with bazedoxifene increased the sialic acids in plasma cells in a similar manner to E2 but did not reach statistical significance. However, we did not detect any alteration in IgG-sialylation with bazedoxifene treatment. Neither estrogen nor bazedoxifene showed any significant alteration in serum protein sialylation but had a minor effect on mRNA expression of glycosyltransferase in the bone marrow, gonadal fat, and liver.

in B cells? E2 and BZA really have activity? It may be inactivated. The given dose of E2 and BZA is optimization? 1. I have required an additional control, non-ovariectomized mice in all experiments.
Answer: This is a good point requested by the editor. Our interest in this paper was to build on the findings from Engdahl et al. Arthritis and Research Therapy 2018 by demonstrating that not only estrogen treatment but also SERM Bazedoxifene (BZA) could inhibit estrogen-deficient loss of sialic acid on IgG, which affects the binding ability to Fc gamma receptors. We could see that estrogen increases the sialic acid on IgG and thereby reduces the pathogenicity, but we could not show that bazedoxifene has the same estrogenic effect. In the paper written by me, Engdahl et al. Arthritis and Research Therapy 2018, I show that estrogen deficiency, OVX, reduced the IgG-Fc sialic acids compared to non-ovariectomized mice, but comparing the estrogen-deficient induction was the scope of this manuscript.  (24)."

Reviewers' comments
Reviewer #1: The paper entitled "Bazedoxifene does not share estrogens effects on IgG sialylation" investigated the effects of bazedoxifene and E2 on IgG sialylation. However, several issues should be addressed:

Major comments
1. The initial description of the abstract is not complete and coherent.
Answer: I appreciate the reviewer bringing this up. The abstract has now been improved and became complete and coherent. We have provided both a clean version and the updated manuscript, along with marked changes. 2. This study requires a control group. Why didn't you study a control group in addition to the Vehicle group?

Answer:
We are uncertain about which type of control has been requested. However, as addressed in the response to the editor, we were only interested to investigate the effect on the post-menopausal status and therefore did not include a sham-operated control. The difference between estrogen deficiency, OVX, and sham was displayed in Engdahl et al. Arthritis and Research Therapy 2018. Here in the present study, we showed that estrogen deficiency, OVX, induced a lower degree of sialic acids on IgG, a phenotype that increased the IgG pathogenicity.

How was the sample size calculated?
Answer: We acknowledge the reviewer's comment. Because this manuscript was submitted to a journal requiring less text, we removed the sample size calculation. We have now included the sample size calculation in the revised manuscript.

Results; line 194-203 " 3.4. Treatment with estrogen and bazedoxifene has minor effects on the mRNA levels of glycosyltransferases in ovariectomized OVA immunized mice:
To determine the impact of E2 and BZA on protein glycosylation enzymes at the mRNA level, we analysed the expression of glycosyltransferases. A slight decrease of B4Galt2 mRNA in bone marrow was observed in the E2-treated group but not with BZA, and this was not shown for either St6gal1 or Fut8 (Fig. 3A). We also found that Fut8 mRNA expression in gonadal fat was significantly reduced with a p-value of p= 0.05 in the E2 treated group, but not that of St6gal1 or B4Galt2 (Fig. 3B). Whereas, in the liver, we did not detect any change in the mRNA expression of St6gal1, B4Galt2, or Fut8 (Fig.   3C)." 6. In the discussion section this sentence needs references: "Consistent with previous studies, we could not find any alteration of sialic acid in total serum protein by any of the treatments, suggesting that sialic acid levels in serum protein are independent of estrogen status." Answer: Thank you for bringing this to our attention. The reference to this sentence has now been added to the revised version of the manuscript.

Discussion: Line 266-269 "This implies that estrogen has an effect on glycosylation.
Consistent with previous studies ( 3, 5), we could not find any alteration of sialic acid in total serum protein by any of the treatments, suggesting that sialic acid levels in serum protein are independent of estrogen status." Result: line 179-181; "The sialic acid on OVA-specific IgG was also investigated but we did not detect any change in either of the treatments ( Supplementary Fig. 2 C)."

Reviewer
Discussion: lines 247-248; "In addition, in this study, neither estrogen nor bazedoxifene, alter the sialylation of OVA-specific IgG." 2. In addition to measure sialation, did the authors check the animals for RA symptoms?
If not, it is important to measure sialation and correlate with RA.
Answer: It would be interesting to look at a RA animal model, but our study was not performed as a RA model, and therefore we did not look for RA symptoms. To understand the pathogenicity of IgG, we needed to massively induce the IgG response, which we did in this experiment with OVA immunization. While a higher dose of BZA may produce a more pronounced effect, it is also likely to lead to alterations in uterine weight. The purpose of using BZA is to engage estrogen's positive effect, such as on bone, without the negative effect of inducing weight in reproductive organs. The aim of this present study was to investigate the ability of BZA to reduce the pathogenic capability of IgG, which could be the basis for future clinical studies in pre-RA patients with autoantibodies and limited clinical signs of RA, as well as the post-menopausal patient from hinder them to develop clinical symptoms.
However, this follow-up study will not investigate the effect of BZA on IgG glycosylation.

Minor:
3. Mention in methods the number of mice/group.

Answer:
Thank you for bringing this to our attention. We have now added the number of mice in the revised version of the manuscript.
Material and method; lines 94-97; "Based on the sample size calculation we started the experiment with 10 mice per group. Due to health concerns, a few mice were taken away during the experiment, and this resulted in n= 8/veh, n=8/E2, and n=8/BZA."